EP0801082A2 - Résines pour la synthèse de peptides en phase solide et leurs procédés de préparation - Google Patents

Résines pour la synthèse de peptides en phase solide et leurs procédés de préparation Download PDF

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Publication number
EP0801082A2
EP0801082A2 EP97111264A EP97111264A EP0801082A2 EP 0801082 A2 EP0801082 A2 EP 0801082A2 EP 97111264 A EP97111264 A EP 97111264A EP 97111264 A EP97111264 A EP 97111264A EP 0801082 A2 EP0801082 A2 EP 0801082A2
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peg
solid
resin
amino
group
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EP0801082B1 (fr
EP0801082A3 (fr
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George Barany
Jane Chang
Nuria A. Sole
Fernando Albericio
Samuel Zalipsky
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University of Minnesota
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University of Minnesota
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/042—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • Spacer arms are essential in many areas of modern biochemistry. Spacer arms can be defined as molecules which link one molecule to another molecule or to an inert support. Polyethylene glycol, for example, has been used advantageously to link enzymes to insoluble carriers and other biomolecules while retaining the activity of the enzyme. M. Stark and K. Holmberg, Biotech. and Bioeng. , 34 :942-950 (1989). This concept has important consequences for industrial processes using immobilized enzymes (e.g., affinity column purification processes), and for diagnostic assays (e.g., ELISA assays). Two other areas in which polyethylene glycol spacer arms have been used are peptide synthesis and sequencing.
  • PEG structures have been used as chemically inert spacer arms because they are compatible with a wide range of solvents. Inman et al., ibid .
  • the use of PEG spacer arms minimizes the steric effects caused by the support.
  • PEG spacer arms provide another useful function in modifying the character of the pore space so that the support-bound reactive moiety is compatible with a wider range of solvents and reagents.
  • PEG-modified polystyrene (PEG-PS) resins have been described for use in solid-phase peptide sequencing. Inman et al., ibid . PEG-PS resins have also been utilized as phase transfer catalysts. W.M. McKenzie et al., J. Chem. Soc. Chem. Commun. , p. 541-543 (1978); S.L. Regen et al., J. Amer. Chem. Soc. , 101 :116-120 (1979); J.G. Heffernan et al., J. Chem. Soc. Perkin II , p. 514-517 (1981); Y. Kimura et al., J. Org. Chem.
  • PEG-PS resins have been described as supports for solid-phase peptide synthesis. Becker et al., Makromol. Chem. Rapid Commun. , 3 :217-223 (1982); H. Hellermann, et al., Makromol. Chem. , 184 :2603-2617 (1983).
  • PEG-PS resins prepared by the referenced methods suffer from several drawbacks. The reactions proceeded poorly with high molecular weight PEG (e.g., greater than 400 daltons) and symmetrical bifunctional PEG tended to form crosslinks.
  • PEG graft copolymers Another method of making PEG graft copolymers is described by Zalipsky et al., In Peptides:Structure and Function , C.M. Deber, V.J. Hruby and K.D. Kopple (eds.), Proc. 9th Am. Pep. Symp., pp. 257-260. Pierce Chem. Co., Rockford, IL (1985).
  • certain heterobifunctional PEG derivatives of defined molecular weight i.e., 2000 to 4000 daltons
  • a method of preparing non-toxic and efficient solid supports which can be used with a wide range of solvents would be valuable for use in solid-phase synthesis or sequencing of peptides or nucleic acids, or for other solid-phase applications.
  • the present invention pertains to polyethylene glycol derivatized graft supports, to methods for making the supports, and to methods of using the supports to synthesize peptides by solid-phase synthesis techniques.
  • the PEG-graft supports of this invention comprise functionalized PEG derivatives which are covalently attached to solid supports.
  • SS represents the solid support.
  • the terminal amino group can optionally be protected by N ⁇ protecting groups, such as Boc, Fmoc and other known protecting groups
  • N ⁇ protecting groups such as Boc, Fmoc and other known protecting groups
  • the core portion of the formula, indicated within the brackets, corresponds to a series of readily available amino-functionalized polyethylene glycol (PEG) polymer derivatives.
  • the resins are constructed such that peptide synthesis can occur at a point other than the terminus of PEG.
  • Such resins are illustrated in Figure 1B and further represented by the general Formula II: wherein Y is a diamino monocarboxylic acid that can optionally be protected by a N ⁇ protecting group; n is an integer from about 5 to about 150; SS is a solid support; A is a straight chain or branched C1-C10 alkyl group, such as methylene, ethylene, propylene, isopropylene, butylene and isobutylene; and R 1 , R 2 and R 3 are independently selected from the group consisting of hydrogen, alkyl groups and aryl groups.
  • the present invention provides several compact, commercially viable routes for making functionalized inert supports for solid-phase applications using monofunctional or homobifunctional polyethylene glycol as the starting material.
  • the present PEG graft supports provide advantageous physical and mechanical properties for solid-phase peptide synthesis, nucleic acid synthesis, and other applications where immobilized molecules are used.
  • Figures 1A and 1B show the general structures of two types of polyethylene glycol-polystyrene (PEG-PS) graft supports.
  • X illustrates the point from which biopolymer chain growth begins.
  • Figure 2 is a schematic showing a series of reactions leading to preparation of a spacer arm linker, its coupling to an amino-functionalized polystyrene resin, attachment of a handle to the resin-bound linker, and its use to synthesize a peptide.
  • Figure 3 is a schematic showing a series of reactions leading to further PEG-PS graft supports.
  • Figure 4 is a schematic showing a series of reactions leading to a PEG-PS graft support in which the biopolymer can be synthesized from the Orn residue.
  • the present invention pertains to resins comprising a functionalized polyethylene glycol derivatives covalently linked to a solid support.
  • the resulting graft support comprises a symmetrical poly(oxyethylene) diamine derivative linked to the support and is represented by Formula I: wherein n is an integer from 5 to about 150; R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently selected from the group consisting of hydrogen (H), and simple alkyl or aryl groups such as methyl, ethyl or phenyl; SS is a solid support; X is H or H 2 N-B-NH-C(O)-A-C(O)-; and A and B are independently a straight chain or branched alkyl group, such as ethylene, propylene, isopropylene, butylene, isobutylene or the like up to C-10 in length (e.g., A derived from succinic, glutaric, adipic or other such acids; B derived
  • One method for producing the graft supports of Formula I is by reacting amino-functionalized core polymers with dicarboxylic acid derivatives, including anhydrides, to produce carboxyl-functional molecules.
  • the diamine polymer is reacted with at least two equivalents of the activated dicarboxylic acid derivative.
  • Dicarboxylic acids which are useful in this method include alkyl diacids having up to about 12 carbon atoms, such as, for example, maleic, succinic, glutaric or adipic acid; anhydrides such as maleic, succinic or glutaric anhydride; or aromatic anhydrides, such as phthalic anhydride.
  • the diamine polymer is reacted with succinic, maleic or glutaric anhydride to make representatives of the claimed compounds, bis(succinylated), bis(maleylated) or bis(glutarylated) PEG.
  • Figures 2 and 3 illustrate the formation and subsequent coupling of these derivatives onto an amino-functionalized solid support.
  • active ester refers to compounds which activate carboxyl groups to make them undergo more ready reactions with amino groups.
  • Activating groups which can be used in the present composition and method include, for example, trichlorophenyl (TCP) esters, pentafluorophenyl (PFP) esters, pentachlorophenyl (PCP) esters and methylphenylpyrazolinone (MPP) esters.
  • TCP trichlorophenyl
  • PFP pentafluorophenyl
  • PCP pentachlorophenyl
  • MPP methylphenylpyrazolinone
  • Symmetrical diamines which can be used as the core portion of the polymer include polymers corresponding to the bracketed portion of the structures shown in Formula I and Formula III.
  • PEG derivatives having a theoretical content of amino groups can be obtained by a number of known processes. See, for example, Duckmann et al., Makromol. Chem. , 182 :1379 (1983); Zalipsky et al., Eur. Polym. J. , 19 :1177 (1983).
  • Polymers which are particularly useful for this purpose include a series of poly(oxyethylene) diamines having a molecular weight up to about 6000 daltons which are commercially available under the tradename Jeffamine® (Texaco Chemical Co., Bellaire, TX).
  • the Jeffamine® poly(oxyethylene) diamine resins are aliphatic primary diamines structurally derived from polypropylene oxide-capped polyethylene glycol. These products are characterized by high total and primary amine contents. Other symmetrical diamines having the desired characteristics can be used. For some applications, symmetrical dicarboxylic acid-functionalized polymers having approximately the same general structure can be used.
  • Carboxyl-functionalized spacer arm linkers produced by the present method are then coupled to appropriate parent carriers which have been functionalized with amino groups.
  • Carriers which are useful as solid phases in the present invention include macromolecules or solids, such as membranes, porous glass, silica, polystyrenes, polydimethylacrylamides, cotton or paper.
  • Solid supports which are particularly useful include amino-functionalized polystyrene, aminomethyl polystyrene, aminoacyl polystyrene and p-methylbenzhydrylamine polystyrene.
  • a particularly preferred support is an amino-functionalized polystyrene-co-1% divinylbenzene.
  • All amino groups of the parent carrier can be covered by reacting one equivalent of the carrier based on the amino groups with an excess of the carboxyl-functionalized derivatives. Most of the amino groups on the carrier become substituted by the polyethylene glycol derivatives, thereby forming spacer arms having one free pendant carboxyl group.
  • an amino functionality on the present PEG derivatives is desirable for many synthesis applications. This can be achieved by acid hydrolysis (see Figure 2) of the amide moiety, thereby exposing the amino group which was originally present on the diamine, or by further coupling of a free or monoprotected low molecular-weight diamine (e.g., ethylene or hexamethylene diamine) with the carboxylate end group (see Figure 3).
  • a free or monoprotected low molecular-weight diamine e.g., ethylene or hexamethylene diamine
  • the terminal pendant maleyl group is selectively hydrolyzed by controlled treatment with an acid, e.g., trifluoroacetic acid or dilute hydrochloric acid (HCl), whereas the other maleyl group, now linking the PEG to the carrier, is essentially stable.
  • an acid e.g., trifluoroacetic acid or dilute hydrochloric acid (HCl)
  • HCl dilute hydrochloric acid
  • the diacid or anhydride is first reacted with the amino-functionalized support, thereby forming an amide-linked carboxyl functionalized support.
  • This carboxyl functionalized support is activated and then contacted with an excess of a bifunctional diamine polymer, represented by the core structure described above.
  • the PEG graft supports described above makes it possible to assemble biopolymers at the PEG terminus as shown in Figure 1A.
  • the resins of the present invention can be constructed such that peptide synthesis can occur at a point other than the PEG terminus.
  • Such resins are illustrated in Figure 1B and are further represented by the general Formula II: wherein Y is a diamino monocarboxylic acid that can optionally be protected by a N ⁇ protecting group; n is an integer from about 5 to about 150; SS is a solid support; A is a straight chain or branched C1-C10 alkyl group, such as methylene, ethylene, propylene, isopropylene, butylene and isobutylene; and R 1 , R 2 and R 3 are independently selected from the group consisting of hydrogen, alkyl groups and aryl groups.
  • a PEG graft support of this type (Formula II) is synthesized using a monofunctionalized polyethylene glycol (e.g., PEG monoamine or PEG monomethyl ether) which has been derivatized.
  • PEG starting materials are either monofunctional Jeffamine® or PEG monomethyl ether which has methoxy and hydroxyl end groups.
  • An amino group on PEG can be reacted with a dicarboxylic acid or anhydride as already described.
  • carboxyl-functionalized derivatives of MPEG can be made by reaction with ethyl bromoacetate, 4-bromovalerate or isocyanacetate, followed by saponification, for example as shown in the equation below.
  • Figure 4 schematically illustrates the synthesis of a PEG resin of Formula II.
  • an N ⁇ -Fmoc, N ⁇ -Boc-Orn-PS resin (or a permutation on this theme) is deblocked selectively, and a monofunctional PEG-acid is coupled on to form a branch orthogonal to the ultimate direction of biopolymer chain growth.
  • An ornithine residue (as shown in Figure 4) is used to link the PEG derivative to the solid support; however, other chemical moieties which have a carboxyl group and two amino groups can be used.
  • PEG-PS Polyethylene glycol-polystyrene graft supports made by the methods of this invention are particularly useful.
  • PEG-PS supports made using the present PEG derivatives have several desirable characteristics for solid-phase applications: they swell in a variety of solvents, are stable under the condition used in most solid-phase synthesis, and behave well in both batch and column reactors used in solid-phase applications, in particular, solid-phase peptide synthesis.
  • Solid-phase peptide synthesis typically begins with covalent attachment of the carboxyl end of a first amino acid to the solid support.
  • the carboxyl group of an N ⁇ -protected amino acid is covalently linked to a handle moiety which is attached to the amino group on the free end (the end not linked to the solid support) of the PEG spacer arm ( Figures 1A, 2 and 3) or at a point other than the PEG terminus ( Figures 1B and 4).
  • a "handle” is defined as a bifunctional spacer which serves to attach the initial amino acid residue to the polymeric support.
  • One end of the handle incorporates a smoothly cleavable protecting group and the other end of the handle couples to the functionalized solid support.
  • Handles which can be used with the present spacer arms in solid-phase peptide synthesis include, for example acid-labile p-alkoxybenzyl (PAB) handles, photolabile o-nitrobenzyl ester handles, and handles such as those described by Albericio et al., J. Org. Chem. , 55 :3730-3743 (1990) and references cited therein, and in co-pending U.S. applications Serial No. 07/576,233 by G. Barany and F. Albericio and Serial No. 07/576,232 by G. Barany, both filed on August 31, 1990, the teachings of all of which are hereby incorporated herein by reference.
  • PAB acid-labile p-alkoxybenzyl
  • the appropriate handles are coupled quantitatively in a single step onto the amino-functionalized supports to provide a general starting point of well-defined structures for peptide chain assembly.
  • the handle protecting group is removed and the C-terminal residue of the N ⁇ -protected first amino acid is coupled quantitatively to the handle.
  • the general synthesis cycle proceeds.
  • the synthesis cycle generally consists of deprotection of the N ⁇ -amino group of the amino acid or peptide on the resin, washing, and, if necessary, a neutralization step, followed by reaction with a carboxyl-activated form of the next N ⁇ -protected amino acid.
  • the present PEG-derivatives are particularly useful as spacer arms, which separate the inert support from the reacting amino acids which are forming the peptide chain during the synthesis process.
  • spacer arm which provides this distance, is a critical parameter in solid-phase applications.
  • PEG spacer arms having an average molecular weight of about 2000 daltons were incorporated between amino functional groups on a polystyrene backbone and the point for attachment of the appropriate handles.
  • the resultant PEG-PS graft supports contained approximately equal weight amounts PEG and PS. These supports shoved reproducible advantage over PS supports with regard to physical and chemical properties such as swelling and in synthesis of model peptides.
  • the PEG-PS supports of this invention allow peptide synthesis to be carried out using acetonitrile as the solvent for all reaction steps and washes. The control experiments using PS and employing acetonitrile as the solvent resulted in no peptide products.
  • PEG-PS graft support was demonstrated by syntheses of a number of large, (e.g., having about 30 to 60 residues) complex peptide sequences, such as cecropin analogues, calcitonin, ⁇ -endorphin, corticotropin releasing factor, two zinc finger binding sequences, and several partial sequences of HIV-1 tat protein.
  • complex peptide sequences such as cecropin analogues, calcitonin, ⁇ -endorphin, corticotropin releasing factor, two zinc finger binding sequences, and several partial sequences of HIV-1 tat protein.
  • the improvements in synthetic efficiency which resulted from use of the present PEG-PS linkers appear to originate from one or more of the following: (i) a spacer arm effect removing the reaction sites from the vicinity of the polymer backbone; (ii) a general environmental effect which modifies the hydrophobic nature of the resin with a concomitantly favorable influence on reaction rates; and (iii) a specific effect on conformationally difficult couplings due to decreased secondary structure (hydrogen bond formation).
  • the PEG derivatives can also be used in nucleic acid synthesis or sequencing as spacer arms or linkers between an inert support and the reacting nucleotide or nucleic acid.
  • the derivatives can be attached to polystyrene resins as described above and used in amidite-mediated DNA synthesis.
  • the PEG-PS resin swells in acetonitrile, which is used as a solvent in the amidite coupling method.
  • Dichloromethane 500 mL was added to extract the polymer, and activated alumina (200 g) was added. After 30 minutes, the organic phase was collected upon filtration, partially concentrated (to ⁇ 100 mL), combined with ethyl ether (700 mL), and brought to -20°C for several hours. The precipitated polymer was collected by filtration, air-dried, and taken up in aqueous sodium hydroxide (1 N, 500 mL) for 3 hours, 25°C. This saponification reaction was quenched by acidification to pH 3 with concentrated aqueous HCl, and the product was extracted into dichloromethane (3 x 250 mL).
  • an aminomethyl polystyrene resin support (0.68 mmol/g) was derivatized with Fmoc-norleucine by standard procedures.
  • a portion of the resultant Fmoc-Nle-resin (0.23 g, 0.55 mmol/g, 0.13 mmol) was deprotected with piperidine-DMF (3:7, v/v) (2 + 10 min) and washed with DMF and dichloromethene.
  • the PEG-diacid from Example 2 (0.86 g, 0.39 mmol, 0.78 mmol carboxyl groups), BOP (88 mg, 0.2 mmol) and HOBt (27 mg, 0.2 mmol) were dissolved in DMF (4 mL), and then a solution of DIEA (45 ⁇ L, 0.26 mmol) in dichloromethane (2 mL) was added. After a 10 min preactivation period at 25°C, the PEG solution was added to the deprotected and washed resin, and coupling was carried out at 25°C for 5 hours to give an essentially negative ninhydrin test.
  • the PEG-functionalized support prepared in Example 4 was washed with trifluoroethanol on a sintered funnel, and transferred to a silanized round bottom flask. A mixture of trifluoroethanol/trifluoroacetic acid/water (8:1:1, 50 mL) was added and the suspension was heated at reflux (oil bath temp. 100°C) and stirred magnetically for 24 hours. The material was then washed with dichloromethane, 5% DIEA in dichloromethane and methanol, and dried in vacuo in P 2 O 5 . Amino group content was determined on an aliquot by picric acid titration, or by loading with an Fmoc-amino acid.
  • the PEG-PS support from Example 5 (0.4 g, 0.12 mmol original Nle sites) was washed with dichloromethane for initial swelling, then DIPCDI (94 ⁇ L, 0.6 mmol) and HOBt (81 mg, 0.6 mmol) in DMF (2 mL) were added to the resin for a 5 minute preactivation period. Ethylenediamine (40 ⁇ L, 0.6 mmol) in DMF (0.5 mL) was then added to the resin for a 5 hour reaction. Final washes with DMF, dichloromethane and methanol were performed.
  • the resin (0.4 g; no weight change from this step) was dried in vacuo over P 2 O 5 , following which the usual loading measurements with Fmoc-Ala revealed a substitution level of 0.11 mmol/g, and an Ala:Nle ratio of 0.30 (consistent with earlier estimate of extent of cross-linking).
  • PEG-PS was prepared according to the procedure set out in Examples 4 and 7, starting with 5 grams of p-methylbenzhydrylamine resin (with loading 0.6 mmol/g), and 8.7 gram of PEG-PS product was obtained (loading 0.17 mmol/g).
  • a 2 gram portion of this PEG-PS was extended with the Fmoc-PAL [5'-(4''-(9-fluorenylmethyloxycarbonyl) aminoethyl-3,5-dimethoxyphenoxy)valeric acid] handle which was linked by the BOP + HOBt protocol (D. Hudson, (1987) J. Org. Chem. , 53 :617-624) to give a totally functionalized ninhydrin negative product.
  • AAAA amino acid analysis
  • HPLC high performance liquid chromatography
  • FAB fast atom bombardment
  • PEG-Orn(Boc)-PS prepared as described in Example 6 was treated with TFA-dichloromethane (1:1) (5 + 25 min) to remove selectively the N ⁇ -Boc group. There followed washing with dichloromethane, neutralization with 5% DIEA in dichloromethane and dichloromethane. Similar to Example 9, an Fmoc-PAL handle was added, and a MilliGen/Biosearch model 9050 peptide synthesizer was used with a DIPCDI + HOBt coupling protocol (0.05 M concentrations), to make the challenging deca-alanyl-valine sequence. In a consecutive run, a normal polystyrene support was used to make the same sequence. The purity of the peptide based on HPLC was 77%, when PEG-PS was used, as compared to 53% when PS was used.
EP97111264A 1990-08-31 1991-08-27 Procédé pour la préparation des résines pour la synthèse de peptides en phase solide Expired - Lifetime EP0801082B1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US57631490A 1990-08-31 1990-08-31
US576314 1990-08-31
US71528991A 1991-06-14 1991-06-14
US715289 1991-06-14
EP95112933A EP0687691B1 (fr) 1990-08-31 1991-08-27 Résines pour la synthèse de peptides en phase solide
EP91916082A EP0546055B1 (fr) 1990-08-31 1991-08-27 Derives de polyethylene-glycol destines a des applications en phase solide

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EP95112933.7 Division 1991-08-27
EP91916082.0 Division 1991-08-27

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EP0801082A2 true EP0801082A2 (fr) 1997-10-15
EP0801082A3 EP0801082A3 (fr) 1998-07-22
EP0801082B1 EP0801082B1 (fr) 2003-04-09

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EP97111264A Expired - Lifetime EP0801082B1 (fr) 1990-08-31 1991-08-27 Procédé pour la préparation des résines pour la synthèse de peptides en phase solide
EP91916082A Expired - Lifetime EP0546055B1 (fr) 1990-08-31 1991-08-27 Derives de polyethylene-glycol destines a des applications en phase solide

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JP3122460B2 (ja) 2001-01-09
EP0546055A1 (fr) 1993-06-16
JP2002080530A (ja) 2002-03-19
EP0687691B1 (fr) 1998-10-07
DE69133235T2 (de) 2003-11-20
DE69120821D1 (de) 1996-08-14
DE69130333D1 (de) 1998-11-12
DE69120821T2 (de) 1997-01-23
EP0687691A3 (fr) 1995-12-27
EP0801082B1 (fr) 2003-04-09
EP0801082A3 (fr) 1998-07-22
DE69133235D1 (de) 2003-05-15
JP3435541B2 (ja) 2003-08-11
DE69130333T2 (de) 1999-03-04
JP2001081099A (ja) 2001-03-27
EP0687691A2 (fr) 1995-12-20
JPH06500331A (ja) 1994-01-13
EP0546055B1 (fr) 1996-07-10
WO1992004384A1 (fr) 1992-03-19
JP3266600B2 (ja) 2002-03-18

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